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Embryos were then fixed in 1% formaldehyde in 1X PBS for 1?h followed by washes in 1X PBS/0.2% saponin. The embryos were blocked in 10% sheep serum (Jackson ImmunoResearch)/1X PBS/0.2% saponin and incubated in ��-DsRed (Clontech) and S46 ( Stainier and Fishman, 1992) antibodies. Embryos were gently compressed under a cover slip and the hearts were photographed with a Zeiss M2BioV12 fluorescent stereomicroscope. Adobe Photoshop was used to count the number of buy AP24534 CMs in each chamber. Only counts from individual experiments were compared to each other because slight variation can occur in CM number between different clutches of embryos. When comparing CM number data, Student's t-test was used to determine if there was a statistically significant difference (pUNC2881 CM counts of the different conditions. CM proliferation was assessed using the Click-iT EdU Kit essentially as has been reported previously (Guner-Ataman et al., 2013?and?Mahler et al., 2010). After EdU incorporation, embryos were processed for immunohistochemistry as described above. Incorporation of EdU in CMs was determined by immunostaining with MF20 (sacomeric myosin) (Stainier and Fishman, 1992). Phospho-Histone H3 (pHH3; Millipore) immunohistochemistry was performed as previously reported (Dohn and Waxman, 2012). 25�C30 embryos at the desired stage were homogenized in TRIzol (Ambion) and then total RNA collected using Purelink RNA Micro Kit (Invitrogen). Genomic DNA contamination was removed using an on-column DNase step. 1?��g RNA was used for cDNA synthesis using the ThermoScript Reverse Transcriptase kit (Invitrogen). Quantitative real time PCR (qPCR) for myl7, vmhc, amhc, nkx2.5, fgf8a, and b-actin was performed using standard PCR conditions in a Bio-Rad CFX PCR machine with Power SYBR Green PCR Master Mix (Applied Biosystems). Expression levels were standardized selleck chemicals to b-actin expression. All experiments were performed in triplicate. Primers sequences for the respective genes are available upon request. To assess CM contribution, Tg(?5.1myl7:EGFP) donor embryos were injected with a mixture of tcf7l1 MOs and rhodamine-dextran or with rhodamine-dextran alone (controls) at the one cell stage. At the sphere stage, ?10�C15 cells from the donor embryos were transplanted into the margin of wild-type host embryos. Embryos were then raised to 48?hpf and scored live for contribution to the heart. To assess the contribution to ACEs, injections were performed as above except that Tg(kdrl:EGFP) embryos were used as donors. Host embryos were scored for their contribution to the ACEs at 48?hpf. To determine if there was a significant difference (p